Genetic regulation of bone mass: from bone density to bone strength

Osteoporosis is a common disease characterized in adults by diminished bone density. Bone is an organ that evolves and grows throughout life, and establishing optimal bone density in childhood and adolescence serves to buffer bone loss later in life. Bone density, a measurable entity, is the clinical substitute for bone strength, or the ability to defend against fracture. Chronic diseases may adversely affect optimal peak bone density. Bone density is under genetic control, as revealed by three lines of investigations. These include (1) the finding of quantitative trait loci for bone density, (2) the finding that specific mutations in genes that are important in the development of osteoblast or osteoclast lineages alter bone density, and (3) the linkeage of known polymorphisms for genes involved in mineral homeostasis to bone density and/or fracture. Future therapeutics for improving peak bone density or delaying bone loss later in life may take advantage of the genetic nature of bone density development.This work was presented in part at the IPNA Seventh Symposium on Growth and Development in Children with Chronic Kidney Disease: The Molecular Basis of Skeletal Growth, 1–3 April 2004, Heidelberg, Germany     

从骨免疫学到骨微生物学：肠道微生物如何调节骨骼

肠道微生物群被称为人体的第二个基因库,在维持人体平衡中起主要作用。不良饮食习惯、抗生素滥用、病理状态和生活环境改变会对肠道菌群产生负面影响,以引起多种疾病。最新研究发现肠道微生物群与骨质疏松症之间有着密切的联系,同时引入了一个新术语"骨微生物学",该研究领域旨在弥合骨骼生理学、胃肠病学、免疫学和微生物学之间的差距。本文简要介绍了肠道菌群通过免疫系统、新陈代谢和内分泌环境以及其他因素影响骨代谢的潜在机制,通过研究肠道菌群与骨骼健康之间的关系,不仅对于维持骨骼健康和最大程度地减少骨质疏松症很重要,而且对于肠道微生物群是否可作为骨质疏松症新型治疗靶标以及是否可用作骨折预测的生物标志物方面具有重要意义。     

Development of bone canaliculi during bone repair

We recently found that silver impregnation staining with protargol (silver protein), that is, a modified Bodian method, is useful for histologically identifying the details of bone canaliculi structure, using thin sections of decalcified bone tissues. With this staining method, we conducted the present study to assess the development of bone canaliculi during the process of intramembranous ossification using a fracture-like stimulation model of the rat femur. After making a drill-hole in the cortex of the rat femur, decalcified thin sections were obtained after 3, 5, 7, and 14 days by the standard paraffin-embedding procedure. Silver staining for bone canaliculi was performed using our previously reported technique. The results showed that woven bone covered the fracture surface of the cortex after 5 days, then immature lamellar bone attached to the woven bone after 7 days, and finally the lamellar bone matured and became thick with appositional growth after 14 days. The osteocytes in the woven bone appeared at an early stage of bone repair and developed a few canaliculi that were short and irregularly distributed in the osteoid matrix, while the osteocytes in the lamellar bone at a late stage formed many bone canaliculi that were long and regularly distributed in mature bone matrix. Therefore, we concluded that woven bone osteocytes may be necessary for induction of the lamellar bone osteocytes followed by active appositional growth of the lamellar bone at the early stage of bone repair, and also that both bone tissues could be clearly distinguished from one another based on the pattern of development of bone canaliculi by the osteocytes, as seen with the use of our sensitive staining method.     

Aneurysmal bone cyst of the occipital bone

A 13 years boy presented with a painless hard and fixed swelling in occipital region for the last three months. Plain X-ray, CT scan and MRI showed an expansile multi loculated cystic lesion in occipital bone. Histopathological examination revealed it to be an aneurysmal bone cyst. Treatment of choice is surgery. However, radiotherapy may be helpful in incompletely excised lesions.     

脱钙冻干异体骨移植对骨缺损的修复作用

目的应用脱钙冻干异体骨（DFDBA）修复骨缺损，解决种植区的骨量不足问题。方法在种植外科中，将脱钙冻干异体骨用于19例种植区骨缺损患者，观察种植体动度、牙齿指数（GI）及x线改变等临床指标，评价其临床应用效果。结果19例植骨手术均获成功，无一例种植体周围炎发生，种植体初期稳定性良好。结论脱钙冻干异体骨既有一定的力学支持能力，又有骨诱导作用，且可塑性强。     

Transmembrane bone matrix gelatin-induced differentiation of bone

In response to chemically-defined bone matrix gelatin (BMG) inside a diffusion chamber implanted in a muscle pouch, mesenchymal cells migrate directionally, aggregate and differentiate into new bone, on theoutside of the chamber. BMG diffuses through double membranes 275 to 300 μm in thickness. The inner membrane of pore size is 0.025 μm and the outer membrane of pore size is 0.45 μm. The inner membrane is 1/20 the pore size and the combination is twice the thickness of membranes previously reported to transfer osteoinductive activity of living cells. Autoradiographs show³⁵S-cysteine-labelled BMG produces very high transmembrane grain counts while³H-proline labelled BMG produces very low transmembrane grain counts. Electron micrographs demonstrate that gelatin-derived, uranyl-acetate-stained fine granules interspersed with ruthenium red-staining coarse granules, diffuse through the membrane of 0.025 μm pore size from the inside out. Solitary pale-staining collagen fibrils, possibly formed in interstitial fluid by renaturation of BMG are found in the interior of the chamber and in the interior of the outer 0.45 μm but not the inner 0.025 μm pore size membrane. Densely-stained new bone collagen fiber bundles cover the outer membrane, fill the 0.45 μm subsurface pores for a depth of 0.20 to 30 μm, and thereby attach the new cartilage and bone deposits to the outer surface of the chamber. BMG powders solubilize rapidly in diffusion chambers and produce high yields of new bone. The relationship between denatured collagen and renatured gelatin fibrils in the process of transfer of the bone morphogen from BMG to mesenchymal cell receptors is an intriguing subject for further investigation.     

Trabecular bone remodeling and bone balance in hyperthyroidism

In vivo tetracycline double-labeled iliac crest bone biopsies from 15 hyperthyroid patients were used for the reconstruction of curves describing the variation of resorption depth and formation thickness with time. The curves emerging were compared to curves reconstructed from 13 age- and sex-matched normal individuals (mean age 44 years). The median function period for resorptive cells in hyperthyroid patients (16 days) was about one-third the resorptive period in normals (51 days). No significant difference between the osteoclast-, mononuclear-, or preosteoblast-like cell resorption depths could be demonstrated between the two groups. Consequently, the median resorption rate in hyperthyroid patients (3.8 μm/day) was more than 3 times higher than the value in the control group (1.1 μm/day). Median Sigma_f, was shorter in the hyperthyroid group (109 days) than in the control group (151 days, P < 0.05), as was the median initial mineralization lag time (5 and 16 days, respectively, P < 0.01). No significant difference between the measured mean completed wall thickness (mcwT) values in the hyperthyroid groups and the control group could be demonstrated (58.1 and 60.5 μm respectively). Median initial mineralization rate in the hyperthyroid group (1.2 μm³/μm² per day) was not significantly higher than the value calculated in the control group (0.9 μm³/ μm² per day), but median initial matrix appositional rate in hyperthyroids (4.8 μm³/μm² per day) was 3 times higher than the value calculated for normals (1.6 μm³/μm² per day) (P < 0.01). Direct measurements of mean completed wall thickness in the hyperthyroid group gave results (58.1 μm) that were not in accordance with the mean completed wall thickness calculated from the growth curve (52.1 μm, P < 0.02). In normals no such discrepancy could be demonstrated. Using the mcwT value estimated from the growth curve, the bone formation period was calculated to 90 days for hyperthyroid patients. This maximal estimate for mcwT was also significantly lower than the mean resorption depth measured in the hyperthyroid group (61.7 μm, P < 0.05), which means that a net negative balance per remodeling cycle existed in the hyperthyroid group. Bone balance was preserved in the control group.     

Repair of long bone defects with demineralized bone matrix and autogenous bone composite

Background:

Repair of diaphyseal bone defects is a challenging problem for orthopedic surgeons. In large bone defects the quantity of harvested autogenous bone may not be sufficient to fill the gap and then the use of synthetic or allogenic grafts along with autogenous bone becomes mandatory to achieve compact filling. Finding the optimal graft mixture for treatment of large diaphyseal defects is an important goal in contemporary orthopedics and this was the main focus of this study. The aim of this study is to investigate the efficacy of demineralized bone matrix (DBM) and autogenous cancellous bone (ACB) graft composite in a rabbit bilateral ulna segmental defect model.

Materials and Methods:

Twenty-seven adult female rabbits were divided into five groups. A two-centimeter piece of long bone on the midshaft of the ulna was osteotomized and removed from the rabbits’ forearms. In group 1 (n=7) the defects were treated with ACB, in group 2 (n=7) with DBM, and in group 3 (n=7) with ACB and DBM in the ratio of 1:1. Groups 4 and 5, with three rabbits in each group, were the negative and positive controls, respectively. Twelve weeks after implantation the rabbits were sacrificed and union was evaluated with radiograph (Faxitron), dual-energy x-ray absorptiometry (DEXA), and histological methods (decalcified sectioning).

Results:

Union rates and the volume of new bone in the different groups were as follows: group 1 - 92.8% union and 78.6% new bone; group 2 - 72.2% union and 63.6% new bone; and group 3 - 100% union and 100% new bone. DEXA results (bone mineral density [BMD]) were as follows: group 1 - 0.164 g/cm², group 2 - 0.138 g/cm², and group 3 - 0.194 g/cm².

Conclusions:

DBM serves as a graft extender or enhancer for autogenous graft and decreases the need of autogenous bone graft in the treatment of bone defects. In this study, the DBM and ACB composite facilitated the healing process. The union rate was better with the combination than with the use of any one of these grafts alone.     

骨搬移技术治疗下肢长骨干慢性骨髓炎伴骨缺损术后骨性愈合不良的原因分析及对策

目的:探讨骨搬移技术治疗下肢长骨干慢性骨髓炎伴骨缺损术后出现骨性愈合不良的原因及其相应对策。方法:回顾性分析2012年6月至2015年12月采用骨搬移技术治疗的38例下肢长骨干慢性骨髓炎伴骨缺损患者的临床资料,男23例,女15例;年龄20~56岁,平均36.5岁;股骨5例,胫骨33例;受伤至骨搬移时间2~19个月,平均7.4个月;骨缺损长度4~12 cm,平均7.3 cm;术后均经过1周"待机期"开始骨搬移,搬移方向由近端向远端30例,由远端向近端3例,双向搬移5例;并及时调整力线及骨搬移速度,患者坚持负重。定期随访,拍摄X线片,观察是否存在搬移间隙矿化成骨不良,对合端不愈合以及再发骨折等并发症;并采用Paley评分标准进行临床评价。结果:38例获得随访,时间12~36个月,平均23.1个月。骨髓炎无复发,但出现多种骨性愈合不良相关的并发症,其中骨搬移过程中发生搬移间隙矿化成骨不良3例,对合端不愈合17例,发生搬移间隙骨折5例,尚未拆除外固定架时发生骨折1例,拆除外固定架后发生骨折4例。带架时间9~27个月,平均16.3个月;外固定指数为1.7~2.7个月/cm,平均2.24个月/cm;根据Paley评分标准评定疗效:骨性结果优12例,良16例,中3例,差7例;功能结果优14例,良18例,中3例,差3例。结论:骨搬移技术有效解决了长骨干骨髓炎伴大段骨缺损的临床难题,但治疗周期长,对预后的影响因素多。因此,应严格适应证选择,同时应细致操作,全程监控,及时随访,并积极指导患者相应的预防策略,以期得到更满意的临床疗效。     

Preservation of bone morphogenetic protein in heat-treated bone.

In operations of bone tumors, reimplantation of resected bone after boiling or autoclaving is a simple means of obtaining both tumor necrosis and skeletal reconstruction. However, such reimplants lose their osteogenesity. We investigated whether bone inductive ability could be maintained in heat-treated bone. Bone morphogenetic protein (BMP) extracted from rabbit bone after heating for various periods at different temperatures was implanted into the muscles of mice to evaluate osteogenetic activity. The maximum new bone formation was observed in specimens treated at 70 degrees C for 10 minutes, followed by those treated at 70 degrees C for 15 minutes. We then measured the temperature in the center of a cortical bone heated in 0.15 N NaCl solution at 50 degrees, 60 degrees, 70 degrees, 80 degrees, and 90 degrees C. Cortical bone center temperature reached that of the surrounding solution within 2.5 minutes. These results indicated that heating at 70 degrees for 10 to 15 minutes was suitable for heat treated-bone to maintain bone inductive ability.     

Vascularized bone graft in carpal bone reconstruction

We report our experience of vascularized bone graft harvested from the volar aspect of the distal radius for carpal bone reconstruction. Thirty cadaveric dissections showed in all cases the volar carpal artery which born from the radial artery. Between 1994 to 2001, we treated 87 scaphoid non-unions with an average follow-up of 41 months (range 6 to 65 months). Union was obtained in 80 patients (92%) with an average delay of 8.6 weeks (range 6 to 24). Between 1994 to 2000 we treated 22 patients with a Kienbock's disease. A radius shortening was always added to the revascularization of lunate by this vascularized bone graft. Preoperative and postoperative MRI was systematically done. The average follow-up was 55 months (range 24 to 92 months). MRI showed healing with good revascularization in 16 cases (74%). Lesions of lunate were stabilized in five cases and we had one failure with secondary palliative procedure. This simple but meticulous technique needs only one approach and allows a sufficient revascularisation.     

Ilizarov bone transport for massive tibial bone defects

This article reports the treatment of massive tibial bone defects by bone transport using the Ilizarov external fixator. Fifteen patients were treated using this technique (3 females and 12 males). The defect size ranged between 7 and 22 cm (average: 10.6 cm). Etiology was infected nonunion in 9 patients, nonunion in 5 patients, and recurrent giant-cell tumor in 1 patient. The affected site was the tibial diaphysis in 10 patients, the lower tibial metaphysis in 4, and the upper tibial epiphysis in 1 patient. The external fixation time ranged from 9 months to 17 months (average: 12.27 months). External fixation index ranged from 21.8 to 42.5 day/cm (average: 35.7 day/cm). There was no recurrence of infection, no recurrence of the tumor, nor fractures after frame removal. We had to graft the docking site in 2 patients for delayed union and 2 patients developed equinus deformity and had tenoplasty for the Achilles tendon at the time of frame removal. Four patients had pin tract infection at > or =1 of the wires and this was successfully treated by antibiotic injection at the wire site. This study suggests that Ilizarov bone transport is a reliable method to fill massive bone defects.     

Repair of large segmental bone defects using bone marrow stromal cells with demineralized bone matrix

Objective: The aim of the present study was to evaluate the effect of tissue‐engineered constructs on repair of large segmental bone defects in goats. Methods: Allogenic demineralized bone matrix (aDBM) was seeded with autologous marrow stromal cells (aMSC) for seven days to construct DBM–MSC grafts prior to implantation. 24 goats were randomly divided into three groups (eight in each). In each group, 3 cm diaphyseal femoral defects were created unilaterally, and subsequently filled with the DBM‐MSC grafts, DBM alone and an untreated control, respectively. Radiological analysis and biomechanical evaluation were performed at 12 and 24 weeks after operation. Results: Obvious increases in radiological scoring and biomechanical strength were found in the DBM‐MSC group when compared to the DBM group. X‐ray examination showed excellent bone healing in the DBM‐MSC group, whereas only partial bone repair was seen in the DBM group, and no healing in untreated controls. Histologically, a tendency to bone regeneration and remodeling was far more obvious for the DBM‐MSC group than the DBM only and untreated controls. Conclusion: Our results strongly suggest that transplantation of bone MSC within a DBM could have advantages for the bone repair of large segmental defects.     

骨搬运治疗四肢大段骨缺损的临床研究

目的 探讨应用骨搬运治疗四肢大段骨缺损的临床疗效.方法 骨搬运治疗四肢大段骨缺损29例,男23例,女6例;年龄18~53岁,平均30岁;骨缺损5~16cm,平均7.5cm.术前均摄双侧肢体等条件X线片,测量并记录骨缺损的长度,对于合并软组织缺损者在清创前后分别记录创面缺损面积.均在截骨术后10~14d开始骨搬运,每天4~6次,每次1/6~1/4mm.记录术后骨痂牵拉时间、骨痂直径率(CDR)、骨愈合时间、支架固定时间及活动功能情况,分析并进行疗效评定.结果 随访18~31个月,平均24个月.18例移植皮瓣全部成活,伤口一期愈合.在骨搬运过程中,因牵拉皮肤导致不适而行皮肤切开调整:15例为一次,10例为两次,4例为三次.骨搬运时间45~158d,平均76.2d.根据CDR值及时调整牵拉速度,22例CDR≥85%,5例80%≤CDR<85%,2例CDR<80%.骨愈合时间5~14个月,平均8.7个月.外固定支架携带时间6~18个月,平均10.1个月.根据Paley等方法评价骨搬运结果及功能.骨搬运结果评价:优25例,良4例;功能评价:优15例,良10,可4例,优良率86.2%.结论 应用Orthofix重建外固定架进行骨搬运是治疗四肢大段骨缺损的有效方法.骨搬运的起始速率为1mm/d,后根据CDR值及时调整,如CDR>85%,可加快为1.2mm/d;如CDR<80%,可减缓为0.5mm/d.     

Long bone nonunions treated with autologous concentrated bone marrow-derived cells combined with dried bone allograft

Introduction

Nowadays the treatment of long bone nonunion continues to be one of the most complex and debated topics due to the large number of failures. For several years, in the relevant literature three factors have been considered essential in the healing process: growth factors and hormones, osteoprogenitor cells (mesenchymal stem cells), and extracellular matrix. The mechanical stability of the fracture site is considered the fourth element of the “Diamond concept theory.” The aim of our study was to evaluate the validity of biological adjuvants of mechanical synthesis allowing a faster healing process of nonunions.

Materials and Methods

We dealt with 19 patients with long bone nonunion. All patients have been treated with concentrated mesenchymal stem cells without bone autologous transplant. We used the Extracell BMC-marrow aspirate protocol of Regen Lab. The radiographic parameters taken into account for the diagnosis of successful healing were the presence of a bridge callus, obliteration of the fracture line and bone cortical continuity. Clinically, the pain was investigated with VAS score (visual analogue scale), where zero means no pain and 10 the worst possible pain.

Results

Radiographic investigation shows complete healing in 78.9 % (15 cases) with an average time to healing of 6.5 months (minimum healing time 80 days) corresponding also in complete remission of clinical symptoms.

Conclusion

The use of growth factors and autologous mesenchymal stem cells through the enforcement of system for tissue regeneration is a valid and innovative biotechnology technique for the treatment long bone nonunions.     

Penetration of acrylic bone cements into cancellous bone

The depth of penetration of five commercial acrylic bone cements into cancellous bone was measured in vitro. Under standard, idealized conditions, cement penetration was found to vary significantly with different cements. Penetration was critically influenced by the coarseness of the cancellous bone and increased directly with the effective volume of the "cells" within the osseous matrix. An inverse correlation was determined between the mean cement viscosity during flow into the bone and final penetration depth. The dough time, set time and working time of each acrylic formulation was found to have no significant effect upon the depth of cement penetration. It is suggested that in addition to the techniques adopted for introduction of cement to the bone, the selection of the bone cement itself may critically influence the incidence of late loosening following total joint replacement.     

Revascularization and new bone formation in heat-treated bone grafts

Human immunodeficiency virus (HIV) infection is one of the possible serious complications associated with bone allografts. In order to prevent infection, grafted bone is sterilized by various treatments. Heat treatment has attracted attention as a simple and practical method. We carried out a histological study of the influence of heat treatment on autogenic bone grafts. To eliminate the problem of antigenicity of grafted bone, we used autografts, not allografts. Three types of heat-treated autografts were employed: heat-treated at 60° C for 30 min, at 80° C for 10 min, and at 100° C for 5 min; as a control, fresh autografts were replaced in the rabbits’ ilium. One, 2, 4 and 8 weeks after grafting, we performed microangiography and prepared two types of samples: transparent and haematoxylin-eosin (H & E) stained. Then, using an image analyzer, we quantitatively measured revascularization and new bone formation in the grafted bone. The grafts heat-treated at 60° C showed early and good revascularization and new bone formation, from 1 to 8 weeks. The grafts heat-treated at 80° C showed relatively good revascularization and new bone formation. However, the grafts heat-treated at 100° C showed unsatisfactory revascularization and bone formation, less than 40% of control 8 weeks after grafting. Therefore, heat treatment at 60–80° C does not seriously affect revascularization and new bone formation. Received: 3 June 1997     

预成纯钛修复体修复下颌骨缺损

目的：探讨应用反求工程和快速原型技术进行下颌骨缺损修复的设计并预制修复体的可行性。方法：应用反求工程和快速成型技术为4名患者完成了下颌骨缺损的修复体设计和制作，并进行了修复体植入手术。结果：修复体就位顺利，各部件达到设计位置，术后患者面部外形满意，咬合关系良好，下颌偏斜纠正。结论：反求结合快速成型能够完成下颌骨缺损的修复体的设计和预制，提高了手术精度，节省了手术时间，在颌骨缺损的个体化和定制化修复中有独特的优势。     

Membranous bone healing and techniques in calvarial bone grafting

Membranous bone grafts have become an integral part of facial skeletal reconstruction. The convenience of harvesting the graft material from a single operative site was no doubt the reason for its initial utilization. In recent years membranous bone has been shown to be more resistive to resorption and perhaps even provide greater strength per unit volume than does its endochondral counterpart. These facts plus a relatively hidden, nonpainful donor site make membranous bone a desirable graft material. Grafts can be harvested in a variety of forms from dust to vascularized segments, totally dependent on the need. The various techniques of harvesting and methods of utilization are discussed. Rigid fixation has enhanced these techniques, and as the technical aspects improve, so do the results. The use of membranous bone has expanded from the field of congenital craniofacial surgery to the correction of traumatic facial deformities to purely aesthetic surgery. As more experience is gained, the utilization and indications for membranous bone grafting will continue to expand rapidly.